Microorganisms that produce useful products through fermentation are known to require a great amount of energy such as ATP (Adenosine 5′-triphosphate) or reducing power such as NADPH (Nicotinamide Adenine Dinucleotide Phosphate) for enhancement of the biosynthetic pathway thereof.
During the metabolism of microorganisms, the intracellular balance of NADH (nicotinamide adenine dinucleotide) used in catabolic reactions and NADPH (nicotinamide adenine dinucleotide phosphate) used in anabolic reactions are very important. The balance is controlled by phosphorylation of NAD or dephosphorylation of NADP as shown in the following formula.NAD++ATP→NADP++ADPNADP+→NAD++phosphate
In E. coli, phosphorylation of NAD is known to be catalyzed by an enzyme called NAD kinase (EC 2.7.1.23) encoded by the nadK (or yfjB) gene. NAD kinase utilizes Mg2+ as a cofactor of an enzymatic reaction, and is inhibited allosterically by NADPH and NADH. It is known that the Km value for NAD+ is 2000 μM, and that for ATP is 2500 μM (Eur. J. Biochem., (2001) 268: 4359-4365).
Dephosphorylation of NADP has rarely been studied in spite of its central importance in the metabolic pathway. Although an NAD kinase homolog in the archaeon Methanococcus jannaschii was shown to have NADP phosphatase activity, genes encoding the enzyme having such activity are not yet identified in eukaryotic and eubacterial sources. In E. coli, the product of the cysQ gene showed high NADP and NADPH phosphatase activities, but kinetic studies of the purified enzyme suggested that it is not the true NADP phosphatase of this organism (Biochem J., (2007) 402:205-218, Biosci. Biotechnol. Biochem., (2008) 72:919-930).
NAD kinase activities are found in many microorganisms, and the NAD-binding site and the active site of NAD kinase that are important for catalytic activity show highly conserved amino acid sequences between species. For example, various microorganisms including Gram-positive bacteria show a high level of homology in the tertiary structure prediction of helices 2, 4, and 5 (each of them is indicated by H2, H4, and H5) (Appl Microbiol Biotechnol (2010) 87:583-593).
NADP generated by NAD kinase finally supplies a reducing power, and in particular, NADP+/NADPH required for mass-production of useful products in E. coli, is an essential element for anabolic reactions (Biochem J., (2007) 402:205-218). In E. coli, NADPH is mainly produced by 1) the oxidative pentose phosphate pathway, 2) NADP-dependent isocitrate dehydrogenase of the TCA cycle (icd gene), and 3) transhydrogenase (pntAB gene) (J Biol. Chem., (2004) 279: 6613-6619).
These reactions produce NADPH using NADP as a substrate, and thus the NADPH level can be increased by increasing the intracellular level of NADP. Therefore, many attempts have been made to increase the intracellular level of NADP for industrial production of various metabolites, for example, 1) NADPH and thymidine production increased by nadK overexpression in E. coli (Biotechnol Lett., (2009) 31:19291936), 2) The amount of NADPH and PHB (polyhydroxybutyrate) production increased by nadK overexpression in E. coli (Appl Microbiol Biotechnol., (2009) 83:939947), and 3) lysine production increased by ppnK overexpression in Corynebacterium, similar to nadK overexpression in E. coli. The key point in all of the above cases is to increase the expression of the nadK gene. However, in each of these cases, a phosphate source such as ATP must also be increased in order to increase the reducing power via the increased NADPH level, resulting from the increased NADP level caused by high expression of NAD kinase.
ATP is mainly produced by an electron transport system or substrate level phosphorylation in microorganisms. Produced ATP is decomposed to supply energy to cells, and reproduced through glycolysis or oxidative phosphorylation. Based on this fact, a study of applying a bacterial ATP regeneration system to a production process has been made in order to supply energy during the mass production of useful products (Biosci Biotechnol Biochem., (1997) 61: 840-845).
However, as described above, there are few studies on the method of increasing a phosphate source, which is required for an increase in the reducing power by high expression of NAD kinase and a subsequent increase of biosynthetic products. In addition, an increase in energy supply via high production of ATP has merely been studied in terms of energy supply to cells, and utilization of ATP as a phosphate source has not been studied in the related art.